The technical field of this invention is recombinant viral vectors and, in particular, recombinant pseudotyped viral vectors, especially recombinant pseudotyped adeno-associated viral (AAV) vectors.
Parvoviridae are small non-enveloped viruses containing single-stranded linear DNA genomes of 4 to 6 kb in length. Adeno-associated virus (AAV) is a member of the parvoviridae family. The AAV genome contains major open reading frames coding for the Rep (replication) and Cap (capsid) proteins. Flanking the AAV coding regions are two nucleotide inverted terminal repeat (ITR) sequences which contain palindromic sequences that can fold over to form hairpin structures that function as primers during initiation of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be necessary for viral integration, rescue from the host genome and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129).
The capsids of parvoviridae have icosahedral symmetry and are about 20-24 nm in diameter. They are composed of three viral proteins (VP1, VP2, and VP3, which are approximately 87, 73 and 61 Kd, respectively) (Muzyczka supra). VP3 represents 90% of the total virion protein; VP2 and VP1 account for approximately 5% each.
AAV can assume two pathways upon infection of a host cell. In the presence of helper virus, AAV will enter the lytic pathway where the viral genome is transcribed, replicated, and encapsidated into newly formed viral particles. In the absence of helper virus function, the AAV genome becomes integrated as a provirus into a specific region of the host cell genome, through recombination between the AAV ITRs and host cell sequences. Specific targeting of AAV viral DNA occurs at the long arm of human chromosome 19 (Kotin et al., (1990) Proc. Natl. Acad. Sci. USA 87:2211-2215; Samulski et al., (1991) EMBO J. 10:3941-3950). This particular feature of AAV reduces the likelihood of insertional mutagenesis resulting from random integration of viral vector DNA into the coding region of a host gene.
The AAV vector has properties that make it unique for gene therapy, for example, AAV is not associated with any known diseases and is generally non-pathogenic. In addition, AAV integrates into the host chromosome in a site-specific manner (See e.g., Kotin et al., (1990) Proc. Natl. Acad. Sci. 87: 2211-2215 and Samulski et al., (1991) EMBO J. 10: 3941-3950). However, clinical trials have indicated that the low transduction rate and low titer of the virus often may limit its use as a therapy in the central nervous system (CNS).
The AAV viral vector uses cellular receptors to attach to and infect a cell. Recently identified receptors include a heparan sulfate proteoglycan receptor as the primary receptor, and either the fibroblast growth factor (FGF), or the integrin aVb5, as secondary receptors (Qing et al. (1999) Nat. Med. 5:71-77 and Summerford et al. (1999) Nat. Med. 5:78-82). Following attachment to the cell, the viral particle undergoes receptor-mediated internalization into clathrin-coated endocytic vesicles of the cell.
Although the AAV viral vectors provide a suitable means for gene delivery to a target cell, they may often display a limited tropism (i.e., the binding and entry of the virus into a cell) for particular cell types. To date, attempts to alter the tropism of AAV vectors have involved introducing a peptide ligand into the capsid coat. For example, Girod et al. introduced a 14 amino acid peptide containing the RDG motif of the laminin fragment P1 into a capsid region of the AAV2 serotype to alter tropism (Girod et al. (1999) Nature Med. 5: 1052-1056). Zavada et al. altered the tropism of an AAV vector by the addition of viral glycoproteins (Zavada et al. (1982) J. Gen. Virol. 63: 15-24). Others have added single chain fragments of variable regions of a monoclonal antibody against CD34 to the N-terminus of the VP2 capsid (Yang et al. (1998) Hum. Gene. Ther. 9: 1929-1937). The major limitation with these approaches is that they require additional steps that covalently link large molecules, such as receptor ligands and antibodies to the virus. This adds to the size of the virus as well as the cost of production. Furthermore, the targeted particles are not homogenous in structure, which may effect the efficiency of gene delivery and transfer. Therefore, a need exists to generate viral vectors with a modified tropism that interact more efficiently with a cell surface. A need also exists for viral vectors with a modified tropism to target cell types that the corresponding wild type virus does not typically target.